Tissue forms derived from membranous tissue

ABSTRACT

The present invention relates generally to tissue forms useful as grafts or implants and derived from membranous tissue. The tissue forms may contain viable endogenous cells. The tissue forms may be sheets or mini sheets, each having quadrilateral, circular, polygonal, or irregular shapes. The sheet tissue form may have a generally quadrilateral shape, with an average length of about 1 to about 20 centimeters and an average width of about 1 to about 20 centimeters. The mini sheet tissue form may have a generally quadrilateral shape, with an average length of about 0.5 to about 9 millimeters and an average width of about 0.5 to about 9 millimeters. A mixture of the mini sheet tissue form and a liquid, such as cryopreservation fluid, saline or buffer solution, is flowable, which means the mixture will pass through a luer-slip tip syringe, or a gauge needle.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional, and claims the benefit, of U.S. patent application Ser. No. 15/867,472, filed Jan. 10, 2018, now abandoned and which claims the benefit of U.S. Provisional Application No. 62/444,653, filed Jan. 10, 2017, the entire disclosures of both of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to tissue forms derived from membranous tissue and useful as grafts. The invention further relates to an apparatus and method for making the tissue forms.

BACKGROUND OF THE INVENTION

Membranous tissues are sheet like and capable of being laid flat. Various membranous tissues exist, including amnion, chorion, umbilical cord, fascia, submucosa, dermis, intestinal, pericardium, peritoneum, and many others. Such membranous tissues are sometimes useful for making tissue forms suitable for use as grafts in various surgical and medical procedures.

For example, amnion and chorion membranes are obtained from donor placental tissue. Tissue forms derived from such membranous tissues can be made into tissue forms that are useful as grafts in various surgical and other medical procedures, such as ophthalmological, orthopaedic, genitourinary, wound healing, burn care, surgical anti-adhesion, and dental, among others.

Tissue forms derived from membranous tissues having various shapes and sizes continue to be developed.

SUMMARY OF THE INVENTION

The present invention relates generally to tissue grafts derived from membranous tissues such as without limitation, one or more of: unseparated placenta (e.g., amniochorion), amnion, chorion, umbilical, fascia, submucosa, dermis, intestinal, pericardium, and peritoneum tissues. The tissue grafts include sheet and mini sheet tissue forms useful as grafts or implants in medical and surgical procedures. The sheet and mini sheet tissue forms have a retained population size of endogenous cells which is at least 70% of an unprocessed population size of the unprocessed membranous tissue from which the tissue grafts are derived.

The present invention relates to a tissue graft comprising at least one processed tissue fragment derived from naturally occurring unprocessed membranous tissue which comprises amnion membrane, wherein the at least one processed tissue fragment has a retained population size of endogenous cells, wherein at least 70 percent of the retained population size of endogenous cells are viable, and wherein the at least one processed tissue fragment comprises either: at least one sheet, each having a shape with an average length of about 1 to about 10 centimeters (cm) and an average width of about 1 to about 10 cm, or at least one mini sheet, each having a shape with an average length of about 0.5 to about 9 mm and an average width of about 0.5 to about 9 mm.

The present invention also relates generally to a method for producing a tissue graft comprising at least one processed tissue fragment derived from naturally occurring unprocessed membranous tissue, the method comprising: obtaining the unprocessed membranous tissue comprising amnion membrane which has an unprocessed population size of endogenous cells; and subjecting the unprocessed membranous tissue to one or more of: a cleaning process, a soaking process, a bioburden reducing process, an additional soaking process, a rinsing process, and a freezing process, thereby producing the at least one processed tissue fragment, which has a retained population size of endogenous cells which is at least 90 percent of the unprocessed population size, and at least 70 percent of the retained population size of endogenous cells are viable, and wherein the at least one processed tissue fragment comprises either: at least one sheet, each having a shape with an average length of about 1 to about 10 centimeters (cm) and an average width of about 1 to about 10 cm, or at least one mini sheet, each having a shape with an average length of about 0.5 to about 9 mm and an average width of about 0.5 to about 9 mm.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further explained with reference to the attached drawings, wherein:

FIG. 1 provides sample images produced by software used to count stained viable and nonviable cells recovered from viable amnion membrane wherein the left image is an unadjusted image of live/dead stained cells and the right image is after applying viable cell hue and brightness threshold limits with a B&W threshold color;

FIG. 2 is a graph showing the calculated cell count for each of three categories of viable amnion membrane, (A) unprocessed, (B) processed according to an exemplary embodiment of the process described herein, and (C1) processed according to a comparative process; and

FIG. 3 is a graph showing the calculated cell count for each of three categories of viable amnion membrane, (A) unprocessed, (B) processed according to an exemplary embodiment of the process described herein, and (C2) processed according to a comparative process; and

FIG. 4 provides representative H&E images of (a) unprocessed amnion, (b) processed viable amnion membrane according to an exemplary embodiment of the process described herein, and (c) processed viable amnion membrane according to a comparative process;

FIG. 5 provides images of positive Alizarin Red S stained (red stain) representative viable amnion membrane samples after culture in osteogenic media for 2 weeks (A), 4 weeks (B), and 8 weeks (C), images of positive von Kossa stained (dark brown/black stain) representative viable amnion membrane tissue cultured in osteogenic media for 2 weeks (D), 4 weeks (E), and 8 weeks (F), as well as images of negative control amnion membrane cultured in cell culture media showing no positive Alizarin Red S (G) and von Kossa (H) staining; and

FIG. 6 provides images of Alcian Blue stained (blue stain) representative viable amnion membrane samples after culture in chondrogenic media for 2 weeks (A), 4 weeks (B), and 8 weeks (C), as well as an image of negative control amnion membrane cultured in cell culture media showing no significant Alcian Blue staining at 8 weeks (D).

DETAILED DESCRIPTION OF THE INVENTION

Detailed embodiments of the present invention are disclosed herein. It should be understood that the disclosed embodiments are merely illustrative of the invention that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the invention is intended to be illustrative, and not restrictive. It should be understood that although various embodiments of the present invention are described below as involving amnion tissue, the invention is equally applicable to other membranous biological tissues, including without limitation, one or more of: unseparated placenta (e.g., amniochorion), amnion, chorion, umbilical, fascia, submucosa, dermis, intestinal, pericardium, peritoneum, and other tissues.

The present invention relates generally to sheet and mini sheet tissue forms derived from naturally occurring membranous tissue and useful as grafts. Membranous tissue includes any tissue that is capable of being laid flat or placed in a planar configuration for processing, even though the tissue may not be flat or planar in its natural or initial state. The membranous tissue may be derived from humans or other mammals. Before processing, the naturally occurring membranous tissues typically have an initial, unprocessed population of endogenous cells.

The sheet tissue form comprises tissue sheets each having quadrilateral, circular, polygonal, or irregular shapes. The thickness of each tissue sheet is the same as the thickness of the membranous tissue or tissues from which it is derived. In some embodiments, each tissue sheet has a quadrilateral shape with an average length of about 1 to about 10 cm and an average width of about 1 to about 10 cm. Accordingly, in some embodiments, the sheet tissue form will comprise tissue sheets that are squares as small as about 1 cm in length and about 1 cm in width, or squares as large as about 10 cm in length or about 10 cm in width, or they may be rectangles or parallelograms having dimensions anywhere in between. The sheet tissue forms comprise tissue sheets each having a thickness from about 25 microns (0.025 mm) to about 1000 micron (1 mm). The sheet tissue form may be combined with a liquid, such as cryopreservation solution, saline or buffer solution, then packaged and stored until use. Where the liquid is cryopreservation solution, the sheet tissue form may be cryopreserved, in some embodiments, after packaging. The sheet tissue form has a population of endogenous cells, a portion of which may be viable endogenous cells. The term “endogenous” as used herein means naturally occurring or present in tissue after harvest and before processing. The term “viable” as used herein refers to having the ability to grow, expand, or develop; capable of living and/or is metabolically active. The tissue form may also contain intact extracellular matrix.

The mini sheet tissue form comprises tissue fragments, or particles, each having quadrilateral, circular, polygonal, or irregular shapes. The thickness of each tissue fragment is the same thickness of the membranous tissue or tissues from which it is derived. Additionally, in some embodiments, each tissue fragment has a quadrilateral shape with an average length of about 0.5 to about 9 mm and an average width of about 0.5 to about 9 mm. Accordingly, in some embodiments, the mini sheet tissue form will comprise tissue fragments that are squares as small as about 0.5 mm in length and about 0.5 mm in width, or squares as large as about 9 mm in length or about 9 mm in width, or they may be rectangles or parallelograms having dimensions anywhere in between. The fragments of the tissue mini sheets each have a thickness from about 25 microns (0.025 mm) to about 5000 micron (5 mm). The mini sheet tissue form may be combined with a liquid, such as cryopreservation solution, saline or buffer solution, then packaged and stored until use. Where the liquid is cryopreservation solution, the mini sheet tissue form may be cryopreserved, in some embodiments, after packaging.

A mixture of the mini sheet tissue form and a liquid, such as cryopreservation fluid, saline or buffer solution, is flowable to which means the mixture will easily pass through a syringe, a needle, or a luer-slip tip. For example, it has been found that a mixture of the mini sheet tissue form and a liquid will easily pass through a a luer-slip tip, a luer-slip tip syringe, or a needle. Furthermore, it has been found that a mixture of the mini sheet tissue form and a liquid will also pass through an 18 gauge needle and sometimes through a 20 gauge needle. The mini sheet tissue form has a population of endogenous cells, a portion of which may be viable endogenous cells.

Amnion Membrane Separation and Processing

The placenta includes an amnion membrane, a chorion membrane and a spongy layer therebetween. The amnion membrane and, consequently, each piece of a tissue form derived therefrom, includes an epithelial layer (closest to the fetus), an underlying basement membrane, a compact layer, and a fibroblast layer (farthest from the fetus and closet to the chorion of the placenta, and to the mother). The compact layer and fibroblast layer together are sometimes referred to as the stromal portion (or side) of the amnion membrane. As described in further detail below, once separated from the placenta and chorion, the amnion membrane is processed (e.g., cleaned to remove blood and blood clots and, optionally, soaked in an antibiotic solution), and then cut into a sheet or mini sheet tissue form. Alternatively, the entire placental membrane, or only the chorion membrane (i.e., without the amnion) could be processed and then cut into the sheet or mini sheet tissue form. Before processing, the naturally occurring amnion tissue typically has an initial, unprocessed population of endogenous cells.

More particularly, the placenta is generally recovered and packaged with sterile salt or buffer solution. Thereafter, the amnion membrane is manually separated from the chorion membrane. The amnion membrane is loosely attached to chorion membrane by the spongy layer which is jelly-like. Thus, the amnion and chorion membranes are easily separated from one another, taking care not to tear the amnion membrane. The separated amnion and chorion membranes may be frozen and stored at temperatures below 0° C. before being thawed and subjected to further processing as described below. At this point, the separated amnion and chorion are considered “unprocessed” tissues having an unprocessed population of endogenous cells, a portion of which are viable.

The amnion membrane is subjected to one or more soaks by contacting the amnion membrane with an isotonic salt solution, having a physiological pH (i.e., from about 7.0 to about 7.4 pH, such as for example about 7.2 pH), for a period of time. For example without limitation, in some embodiments, Hank's balanced salt solution (HBSS) may be used as the isotonic salt solution. The isotonic salt solution may also be a buffer solution. In some embodiments, the period of time for soaking is from about 5 to about 60 minutes. In other embodiments, the period of time for soaking may be up to about 2 hours, or up to about 4 hours, or up to about 6 hours, or up to about 12 hours or even up to about 24 hours. In some embodiments, the soaking is performed for a period of about 10 to about 20 minutes. In some embodiments, a first soak is performed for a period of about 10 to about 20 minutes, followed by draining the isotonic salt solution away from the amnion membrane, and then a second soak is performed with fresh isotonic salt solution for a period of about 10 to about 20 minutes. During or in between the soaks, the amnion membrane may be manually manipulated and cleaned to remove blood, blood clots from the amnion membrane. In some embodiments, soaks for periods of time different than mentioned above are performed. In some embodiments, additional soaks are used.

Optionally, the soaked and cleaned amnion membrane may be contacted or rinsed with antibiotic solution to minimize the bioburden of the amnion membrane. The antibiotic solution may, for example without limitation, comprise one or more of vancomycin, gentamicin, primaxin, and amphotericin B, or antibiotics from these families. The amnion membrane and antibiotic solution may be agitated to facilitate loosening and separation of bioburden, such as bacteria and other microbes, from the amnion membrane. To rinse away the antibiotic solution, one or more soaks are performed. The period of time for these soaks is the same as described above for the earlier soaks. In some embodiments, for example without limitation, after rinsing with an antibiotic solution, two soaks using Hank's balanced salt solution (HBSS) for a period of about 5 to about 10 minutes each may be performed, followed by a third soak using HBSS for a period of about 30 to about 40 minutes. In some embodiments, soaks for different periods of time than mentioned above are performed. In some embodiments, additional soaks are used.

The resulting processed amnion membrane is a translucent, sometimes clear, membrane sheet with some elasticity, an irregular shape and irregular edges. The degree of translucency and the shape of the amnion membrane will vary with the different donors. The amnion membrane contains live viable cells including, without limitation, amniotic epithelial cells (AEC) and amniotic mesenchymal stromal cells (AMSC). The AECs are typically found in the epithelial layer of the amnion membrane and the AMSCs are typically found in the stromal layer of the amnion membrane. The foregoing processing method preserves the viability of the AECs and AMSCs. After processing as described above, the amnion membrane tissue has a retained population of endogenous cells, a portion of which are viable.

In preparation for cutting the amnion membrane with one or more blades to produce the amnion tissue forms, any remaining liquid may be drained from the processed amnion membrane. The amnion membrane is then laid flat on a cutting surface, such as a cutting block or other planar surface capable of withstanding pressure from cutting blades of the cutting apparatus. The amnion membrane is laid flat with the epithelial side down and in contact with the planar surface, and the stromal side facing upward and exposed. Small cuts or slits may be made in the amnion membrane to ensure that it lies flat on the planar surface, without wrinkles or folds and without air between the membrane and planar surface. Optionally, the upward-facing stromal side of the amnion membrane is manually blotted with wetted wipes to further remove excess liquid, which helps prevent the amnion membrane from sticking to the cutting blades, such as due to surface tension. In some embodiments, depending on the material of which the planar surface is made, the amnion membrane may instead be laid flat with the epithelial side up and the stromal side down.

The amnion sheet or mini sheet tissue forms comprising quadrilateral shaped sheets or fragments, respectively, of amnion which may contain viable cells. In some embodiments, each amnion particle is a square having a width of about 2 mm and a length of about 2 mm and the tissue form is an amnion mini sheet tissue form. Furthermore, the amnion mini sheet tissue form contains about 300,000 to about 900,000, such as from about 500,000 to about 700,000, combined AEC and AMSC cells per square centimeter (cm²) equivalent of amnion mini sheet tissue form. The cell count was performed on histological cross sections of amnion tissue of known section thickness, with separate counts for AEC and AMSC cells and extrapolated from the actual counts to the above-reported equivalent counts. The total cell count is the total of live and dead cells present in the tissue.

The amnion sheet or mini sheet tissue forms produced by the methods described and contemplated herein retain a greater number of cells compared to the natural unprocessed amnion membrane from which they are derived than amnion tissue forms prepared by other processes such as, without limitation, those described in each of U.S. Pat. No. 8,980,630 to Woodbury, et al., and U.S. Patent Application Publication No. 2015/0010610 to Tom, et al., the disclosures of which are hereby incorporated herein in their entireties. The methods described and contemplated herein differ in various respects from those disclosed in each of the aforesaid references. For example without limitation, the methods described and contemplated herein are characterized in that they include one or more of the following features:

-   -   do not involve use of any solutions containing anticoagulants,     -   do involve use of one or more solutions that contain one or more         monosaccharides such as, without limitation, glucose;     -   do involve rinsing with one or more antibiotic solutions for         relatively short periods of time such as, without limitation, at         least 1 minute and less than about 60 minutes, or less than         about 40 minutes, or less than about 30 minutes, or less than         about 20 minutes, or less than about 10 minutes, or less than         about 5 minutes;     -   do involve one or more freezing steps performed at a controlled         freezing rate;     -   do involve use of one or more cryopreservation solutions lacking         serum.

Processing and Packaging of the Amnion Mini Sheet Tissue Form

Once the amnion tissue form comprising amnion sheets or mini sheets has been produced, such as by using a cutting apparatus, a predetermined quantity of the amnion tissue form is measured and delivered to a container. Then, a predetermined amount of a liquid, such as saline, buffer solution or any available cryopreservation solution, is combined with the amnion tissue form in the container.

Where the amnion tissue form is a sheet tissue form comprising amnion tissue sheets, the predetermined quantity may, for example without limitation, consist of a single amnion sheet. Alternatively, in some embodiments, the predetermined quantity may be an amount that includes more than one, or even a plurality (i.e., three or more) of, the amnion tissue sheets. In some embodiments involving the amnion sheet tissue form, the container used may be a packaging system particularly designed to contain and store such tissue forms prior to use. See, for example without limitation, the packaging system described in the patent application entitled “Packaging System For Tissue Grafts,” having U.S. Ser. No. 15/402,806, filed Jan. 10, 2017, and issued as U.S. Pat. No. 10,695,157 on Jun. 30, 2020, and hereby incorporated by reference herein in its entirety. In some embodiments, the container may be any container capable of retaining the sheet tissue form and liquid solution for a period of time prior to use.

In some embodiments which involve the mini sheet tissue form, the liquid may, for example, be combined with the amnion mini sheet tissue form in a weight ratio of at least 10:1 of liquid to amnion mini sheet tissue form. The liquid may, for example, be combined with the amnion mini sheet tissue form in a volumetric ratio of at least 5:1 of liquid to amnion mini sheet tissue form. The liquid may, for example, be combined with the amnion mini sheet tissue form in a volumetric ratio of at least 3:1 of liquid to amnion mini sheet tissue form. The liquid may, for example, be combined with the amnion mini sheet tissue form in a volumetric ratio of at least 1:1 of liquid to amnion mini sheet tissue form. The liquid may, for example, be combined with the amnion mini sheet tissue form in a volumetric ratio of at least 1:3 of liquid to amnion mini sheet tissue form. The liquid may, for example, be combined with the amnion mini sheet tissue form in a volumetric ratio of at least 1:5 of liquid to amnion mini sheet tissue form. The liquid may, for example, be combined with the amnion mini sheet tissue form in a volumetric ratio of at least 1:10 of liquid to amnion mini sheet tissue form. In some embodiments, for example, a 7.5 ml quantity of the amnion mini sheet tissue form is combined in a 50 ml graduated cylinder with 75 ml of cryopreservation solution (e.g., 10% by volume dimethyl sulfoxide (DMSO), or Dulbecco's Modified Eagles Media (DMEM), Fetal Bovine Serum and DMSO in a 60/30/10 percent by volume ratio). In some embodiments, for example, a 7.5 ml quantity of the amnion mini sheet tissue form is combined in a 50 ml graduated cylinder with 37.5 ml of cryopreservation solution (e.g., 10% by volume dimethyl sulfoxide (DMSO), or Dulbecco's Modified Eagles Media (DMEM), Fetal Bovine Serum and DMSO in a 60/30/10 percent by volume ratio). In some embodiments, for example, a 7.5 ml quantity of the amnion mini sheet tissue form is combined in a 50 ml graduated cylinder with 22.5 ml of cryopreservation solution (e.g., 10% by volume dimethyl sulfoxide (DMSO), or Dulbecco's Modified Eagles Media (DMEM), Fetal Bovine Serum and DMSO in a 60/30/10 percent by volume ratio). In some embodiments, for example, a 7.5 ml quantity of the amnion mini sheet tissue form is combined in a 50 ml graduated cylinder with 7.5 ml of cryopreservation solution (e.g., 10% by volume dimethyl sulfoxide (DMSO), or Dulbecco's Modified Eagles Media (DMEM), Fetal Bovine Serum and DMSO in a 60/30/10 percent by volume ratio). In some embodiments, for example, a 7.5 ml quantity of the amnion mini sheet tissue form is combined in a 50 ml graduated cylinder with 2.5 ml of cryopreservation solution (e.g., 10% by volume dimethyl sulfoxide (DMSO), or Dulbecco's Modified Eagles Media (DMEM), Fetal Bovine Serum and DMSO in a 60/30/10 percent by volume ratio). In some embodiments, for example, a 7.5 ml quantity of the amnion mini sheet tissue form is combined in a 50 ml graduated cylinder with 1.5 ml of cryopreservation solution (e.g., 10% by volume dimethyl sulfoxide (DMSO), or Dulbecco's Modified Eagles Media (DMEM), Fetal Bovine Serum and DMSO in a 60/30/10 percent by volume ratio). In some embodiments, for example, a 7.5 ml quantity of the amnion mini sheet tissue form is combined in a 50 ml graduated cylinder with 0.75 ml of cryopreservation solution (e.g., 10% by volume dimethyl sulfoxide (DMSO), or Dulbecco's Modified Eagles Media (DMEM), Fetal Bovine Serum and DMSO in a 60/30/10 percent by volume ratio).

The amnion mini sheet tissue form and liquid mixture may then be divided into smaller amounts and combined with additional liquid in smaller containers suitable for individual packaging. In some embodiments, for example, a 0.05 ml sample of amnion mini sheet tissue form mixture (i.e., amnion fragments and cryopreservation solution) is combined with 0.5 ml of cryopreservation solution in a vial capable of withstanding a cryopreservation process. In some embodiments, for example, a 0.075 ml sample of amnion mini sheet tissue form mixture is combined with 0.40 ml of cryopreservation solution in a vial capable of withstanding a cryopreservation process. In some embodiments, for example, a 0.10 ml sample of amnion mini sheet tissue form mixture is combined with 0.30 ml of cryopreservation solution in a vial capable of withstanding a cryopreservation process. In some embodiments, for example, a 0.15 ml sample of amnion mini sheet tissue form mixture is combined with 0.35 ml of cryopreservation solution in a vial capable of withstanding a cryopreservation process. In some embodiments, for example, a 0.30 ml sample of amnion mini sheet tissue form mixture is combined with 0.45 ml of cryopreservation solution in a vial capable of withstanding a cryopreservation process. In some embodiments, for example, a 0.60 ml sample of amnion mini sheet tissue form mixture is combined with 0.40 ml of cryopreservation solution in a vial capable of withstanding a cryopreservation process. In some embodiments, for example, a 1.0 ml sample of amnion mini sheet tissue form mixture is combined with 0.50 ml of cryopreservation solution in a vial capable of withstanding a cryopreservation process. Alternatively, the amnion mini sheet tissue form mixture may be combined with the liquid prior to being placed into a container.

In embodiments where the liquid combined with the sheet or mini sheet tissue form is a cryopreservation solution, the containers of mixtures of amnion sheet or mini sheet tissue form and cryopreservation solution are then sealed into individual packages. The amnion tissue form may then be cryopreserved, in the containers and packaging, using a controlled rate freezer at a cooling rate that maintains the viability of the cells in the mini sheet amnion tissue form. Cryopreservation temperatures may range from less than about 0° C. to about −196° C., for example without limitation, about −20° C., about −40° C., about −50° C., about −60° C., about −70° C., about −80° C. or about −100° C. In some embodiments, the controlled cooling rate is about <=2° C. per minute, and is permitted to ramp up to <=5° C. per minute toward the end of the cryopreservation process since the amnion tissue form already frozen by that time. In some embodiments, the packages are stored frozen at vapor phase liquid nitrogen tank temperatures, ultra-low freezer temperatures, or freezer temperatures until use. In some embodiments, the packages are stored at refrigeration temperatures, such as from about 1 to about 10° C.

The amnion sheet and mini sheet tissue forms containing viable cells are suitable, for example without limitation, for use as a wound or tissue covering to cover ulcers, burns, or other wounds. They may also be used, for example without limitation, to cover interior wounds such as tunneling wounds or surgical wounds. The amnion sheet and mini sheet tissue form are thawed and rinsed or diluted prior to application to reduce the concentration of cryoprotectant agent. The amnion sheet and mini sheet tissue form are also capable of multipotential differentiation which could include chondrogenic differentiation, adipogenic differentiation, osteogenic differentiation and mineralization, all of which may be useful properties in potential orthopaedic or other surgical applications.

In some embodiments, the amnion (sheet or mini sheet) tissue form may not contain viable cells (i.e., the cells have been killed and/or removed). In such embodiments, the packaged amnion sheet tissue form may be stored at deep freeze temperatures such as from about −180° C. to about −40° C. with or without cryopreservation solution (i.e., another liquid could be added to the amnion tissue form at the time of packaging). Alternatively, in such embodiments, the packaged amnion tissue form may be stored at frozen temperatures such as from about −30° C. to about 0° C. with or without cryopreservation solution. Alternatively, in such embodiments, the packaged amnion tissue form may be stored at refrigeration temperatures such as from about 0° C. to about 8° C. with or without cryopreservation solution. Alternatively, in such embodiments, the packaged amnion tissue form may be stored at ambient temperatures such as from about 10° C. to about 30° C. with or without cryopreservation solution.

In some embodiments where the amnion sheet or mini sheet tissue form does not include viable cells (i.e., the cells have been killed and/or removed), the packaged amnion (sheet or mini sheet) tissue form may be lyophilized with cryopreservation solution, as described above for the viable amnion tissue forms. Alternatively, in such embodiments, the packaged amnion tissue form may be lyophilized without cryopreservation solution, as described above for the viable amnion tissue forms. Alternatively, in such embodiments, the packaged amnion tissue form may be air-dried without vacuum, as described above for the viable amnion mini sheet tissue form. Alternatively, in such embodiments, the packaged amnion tissue form may be air-dried with vacuum, as described above for the viable amnion tissue form. Alternatively, in such embodiments, the packaged amnion tissue form may be heat-dried without vacuum, as described above for the viable amnion tissue form. Alternatively, in such embodiments, the packaged amnion tissue form may be heat-dried with vacuum, as described above for the viable amnion tissue form.

It will be understood that the embodiments described herein are merely exemplary and that a person of ordinary skill in the art may make many variations and modifications without departing from the spirit and scope of the invention. For example, in some embodiments, the membranous tissue used to produce the sheet or mini sheet tissue forms may be unseparated amnion and chorion membranes (i.e., an unseparated placenta), or amnion and chorion membranes that have been separated and then re-layered on one another, or umbilical cord tissue that is intact or has been cut lengthwise so as to be capable of being laid flat. Any of these tissues (whole placenta, amnion-chorion combination, chorion, umbilical cord) may be processed and cut in a manner substantially similar to that described above for amnion tissue alone. In fact, as will be apparent to persons of ordinary skill in the relevant art, any of the suitable membranous tissues may be processed and cut substantially as described above, or with appropriate changes to the above-described processing methods. All such variations and modifications are intended to be included within the scope of the invention, as defined by the appended claims.

EXAMPLES Methodology

In all of the following examples, samples of amnion tissue were tested to determine total cell content and what percentage of those were viable cells. The following description explains the method used for this testing which involved enzymatic tissue digestion, separation of cells from tissue remnants, followed by Trypan Blue and live/dead staining of the collected cells and the visualization and quantification of the stained cells.

Enzymatic tissue digestion is the use of enzymes and chemicals to detach and/or recover cells from intact tissue. Trypan Blue staining is a method utilizing Trypan Blue, a cell membrane-impermeable dye, to label nonviable cells that have compromised cell membrane integrity. Live/Dead Staining is a method to fluorescently label cells (i.e. using a commercially available kit) to distinguish between viable and non-viable cells.

List of Materials and Equipment Used:

-   -   Collagenase Type II 1 gram (Worthington Biochemicals, Cat.         LS004196)     -   0.25% Trypsin EDTA 1×(Mediatech, Cat. 25-053-CI)     -   Lactated Ringers and 5% Dextrose Saline Injection, USP     -   Dulbecco's Modification of Eagle's Medium/Ham's F-12 50/50 Mix         without phenol red (Mediatech, Cat. 16-405-CV)     -   Dulbecco's Modification of Eagle's Medium/Ham's F-12 50/50 Mix         with phenol red (Mediatech, Cat. 10-092-CV)     -   Dulbecco's Phosphate-Buffered Saline, 1×(Mediatech, Cat.         21-031-CM)     -   Penicillin-Streptomycin Solution, 100×(Mediatech, Cat.         30-002-CI)     -   Fetal Bovine Serum, Premium (Heat Inactivated) (Mediatech, Cat.         35-016-CV or equivalent)     -   Corning glutaGRO, Liquid (Mediatech, Cat. 25-015-CI)     -   Trypan Blue stain solution (MP Biomedical, Cat. ICN1691049 or         equivalent)     -   LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, Cat. L3224)     -   CO₂ Incubator (NuAire)     -   6-well; Standard tissue culture; Flat-bottom (Fisher Scientific,         Cat. 08-772-1B)     -   100 μm cell strainer (Fisher Scientific, Cat. 22-363-549)

Tissue Thaw and Equilibration:

Supplemented DMEM/F12 media was prepared by adding 10% FBS, 1% PenStrep solution, and 1% glutaGRO solution final v/v to DMEM/F12 media with phenol red (i.e. 10 mL FBS, 1 mL PenStrep solution, 1 mL glutaGRO solution to 88 mL of DMEM/F12). The prepared media was stored at 4° C. in the dark. Immediately before use, the prepared media was warmed in a 37° C. water bath.

Each frozen 5 cm×5 cm viable amnion sheet sample was removed from its outer Tyvek pouch, but retained in its inner pouch and submerged in a bath of at least 1 L of room temperature water until fully thawed (i.e., no ice is visible inside pouch) for up to 15 minutes. Each sample was removed from its inner pouch but kept in its retainer transferred into a bath of at least 200 mL of room temperature “Lactated Ringers and 5% Dextrose Saline Injection, USP,” then submerged for 5 minutes to rinse out DMSO. Each 5 cm×5 cm tissue sheet was placed into a separate well of a 6 well tissue culture plate, to which 5 mL of supplemented DMEM/F12 culture media was added. Each 5 cm×5 cm sheet was allowed to equilibrate in media overnight in a CO₂ incubator at 37° C. and 5% CO₂ level.

Enzymatic Tissue Digestion:

Prepare 0.75% collagenase II solution by adding 1 g of powdered collagenase II per 133 mL of DMEM/F12 media without phenol red. Warm the collagenase II solution and 0.25% trypsin solution in a 37° C. water bath immediately prior to use.

Each 5 cm×5 cm viable amnion sheet sample was placed into a separate 50 mL conical tube containing 40 mL of collagenase II solution. Each conical tube was capped and laid sideways on an incubator shaker at 65 rpm for 40 minutes at 37° C., and then centrifuged at 2000 rpm for 10 minutes at ambient temperature. Taking care not to aspirate tissue, liquid in each tube was aspirated down to 5 mL remaining, and then 40 mL of 0.25% trypsin was added. Each conical tube was recapped and laid sideways on an incubator shaker at 65 rpm for 15 minutes at 37° C. After shaking, 5 mL of Fetal Bovine Serum was added to each tube to neutralize the trypsin.

Each tube's contents was poured through a separate 100 μm cell strainer and into a new 50 mL conical tube, to remove remaining tissue and debris. The new conical tubes were centrifuged at 2000 rpm for 10 minutes at ambient temperature. The liquid in each tube was aspirated down to 5 mL remaining, and then the last 5 mL of solution was transferred into a 15 mL conical tube. The 15 mL conical tubes were centrifuged at 2000 rpm for 10 minutes at ambient temperature. The supernatant from each conical tube was aspirated out of each tube and 1 mL of DMEM/F12 without phenol red was added to each. The contents of each tube was pipetted up and down for a minimum of 10 times to resuspend the cells, until no visible cell pellets were visible.

A sample of the cell suspension was obtained for Trypan Blue staining, as described below. The remainder of each cell suspension was transferred into a separate microcentrifuge tube for performing live/dead staining per, as also described below.

Trypan Blue Staining and Cell Count:

Preparing a stained cell suspension was performed by combining 50 μL of each cell suspension with 50 μL of Trypan Blue in a microcentrifuge tube. The stained cell suspension sat briefly (approximately 1-2 minutes) before transferring 10 μL of it into a hemocytometer having four quadrants. Viable and non-viable cells were manually counted in each of the four quadrants of the hemocytometer, taking care to note that the cells were more rounded in shape than debris, which were more irregular and typically smaller in size. Non-viable cells stained blue due to Trypan Blue dye penetration into the cell. Viable cells remained unstained and appeared clear/white. As long as there is no blue color inside the cell, even cells having black specks were counted as viable. The Total Cell Count was calculated for each suspension (total digested cells per sample) by using the following hemocytometer conversion formula with a Dilution Factor of 2 and a Total Volume of 1 mL:

${{Total}\mspace{14mu} {Cell}\mspace{14mu} {Count}} = {\frac{{{Viable}\mspace{14mu} {Cell}\mspace{14mu} {Count}} + {{Nonviable}\mspace{14mu} {Cell}\mspace{14mu} {Count}}}{4}*\left( {{Dilution}\mspace{14mu} {Factor}} \right)*\left( {{Total}\mspace{14mu} {Volume}} \right)*\frac{10,000\mspace{14mu} {cells}}{m\; L}}$

When cells were too dense in the hemocytometer to count easily, a new sample of cell suspension was taken and further diluted in DMEM/F12 without phenol red, followed by repeating the aforesaid steps starting with preparing a new stained cell suspension. When necessary, the dilution factor was adjusted in cell count calculation.

Live/Dead Staining and Cell Count:

Because the live/dead stain is light sensitive, all live/dead staining steps were performed in the dark. Each working solution of the live/dead stain was prepared on the day it was used, as follows. The reagents were thawed at room temperature. 20 μL of 2 mM ethidium homodimer-1 (EthD-1) was added to 10 mL Dulbecco's Phosphate-Buffered Saline (DPBS) to form an EthD-1 solution, which was vortexed to mix. 5 μL of 4 mM calcein AM was added to the EthD-1 solution and vortexed to mix. The working solution (4 μM EthD-1, 2 μL calcein AM) was kept in foil to avoid exposing the solution to light. In some cases, rather than preparing the live/dead stain from reagents, as described above, a live/dead viability kit (LIVE/DEAD® Viability/Cytotoxicity Kit, from Invitrogen) was used to provide the live/dead working solution applied to the sample of enzymatically digested cell suspension, as follows.

Each microcentrifuge tube containing enzymatically digested cell suspension was centrifuged in a microcentrifuge for 6 minutes at 300×g. The resulting supernatant was removed by aspirating. 1 mL of the live/dead working solution was added to each microcentrifuge tube, followed by pipetting up and down to resuspend cells. The microcentrifuge tubes were incubated in the dark for 20 minutes at room temperature.

The tubes were centrifuged for 6 minutes at 300×g, supernatant was removed from each tube by aspirating, followed by adding 1 mL of fresh DPBS to each microcentrifuge tube and pipetting up and down to resuspend cells again. The foregoing centrifuge-aspirate-pipette cycle was repeated twice more, for a total of three cycles. 50 μL of each cell suspension was pipetted onto a separate microscope slide and carefully covered with a microscope coverslip, avoiding creating air bubbles.

Because the live/dead stain is light sensitive, the following live/dead cell enumeration steps to produce a representative image for each sample are performed in the dark. Cells on each slide are visualized using a dual FITC/TRITC filter on the microscope, using a 2-second manual exposure. EthD-1 (indicating dead cells) was red and calcein AM (indicating live cells) was green. Each coverslip was divided evenly into multiple smaller sampling areas (for example, as shown below) and a representative image taken from each area.

1 2 3 4 5 6 7 8 9

A public domain, Java-based image processing program known as IMAGEJ Version 1.45 s was used to analyze each representative image produced by the foregoing steps and determine the number of viable cells and nonviable cells. IMAGEJ was obtained from the National Institutes of Health, of Bethesda, Md., USA. The software was downloaded from the NIH website at https://imagej.nih.gov/ij/.

Using IMAGEJ, for each representative image, the image was opened and a conversion scale applied by drawing a line over the scale bar and going to Analyze->Set Scale. The known distance and units were entered. The “Global” option was checked to set a global scale, which applied a size limit for particle analysis at a later step. Menu options Image->Adjust->Threshold were successively selected. A hue filter (50-255 pass) and a brightness filter (150-255 pass) with B&W threshold color to filter out low intensity and/or red stains were selected/applied. Menu options Analyze->Analyze Particles were successfully selected. A size limit of 75-800 um² was applied to filter out small artifacts and larger debris and then the “Summarize” option was checked. FIG. 1 shows how the foregoing steps in IMAGEJ change the representative image of live/dead stained cells recovered from viable amnion membrane. More particularly, the left image provides an unadjusted image of live/dead stained cells and the right image is after applying viable cell hue and brightness threshold limits with a B&W threshold color with the IMAGEJ software.

For each representative image processed as described above using IMAGEJ, the number of viable cells as indicated by a relatively strong green fluorescence with a stain size appropriate for a cell were reported. The nonviable cells were either manually counted, or the foregoing procedure was repeated for red stains (i.e., 0-50 pass instead of 50-255 pass hue filter, and 10-800 um{circumflex over ( )}2 particle size). However, whenever possible, nonviable cells were manually counted because automated counts by IMAGEJ were known to sometimes return false positives because EthD-1 stains nuclei, not whole cells, and the stained nuclei are closer in size to non-cellular artifacts. The % Viable Cells for each representative image was calculated as follows:

$\frac{{Viable}\mspace{14mu} {Cells}}{{{Viable}\mspace{14mu} {cells}} + {{Nonviable}\mspace{14mu} {Cells}}}$

Example 1

Cell viability in viable amnion tissue samples was investigated by the methodology described above as applied to amnion cells recovered from cryopreserved samples of three different donors using a live/dead staining analysis.

Method of Viable Amnion Tissue Preparation:

The viable amnion tissue samples were prepared as follows. A fresh placenta was obtained. The amnion membrane was manually separated from the chorion and cut free from the placenta.

The amnion was placed into a first basin with HBSS for 5-10 minutes and manually cleaned during this period with wetted wipes to remove blood and blood clots. The amnion was transferred into a second basin with HBSS for 10-15 minutes and manually cleaned during this period with wetted wipes to remove blood and blood clots.

The amnion was then transferred into a flask containing antibiotic solution comprising vancomycin (50 μg/mL), gentamicin (50 μg/mL), amphotericin B (2.5 μg/mL), and HBSS. The flask was placed on an orbital shaker and agitated at 65 rpm for 60 minutes.

The amnion was then transferred to a second flask containing HBSS, placed onto an orbital shaker, and agitated at 65 rpm for 5 minutes. The amnion was then transferred to a third flask containing HBSS, placed onto an orbital shaker, and agitated at 65 rpm for 5 minutes. The amnion was then transferred to a holding basin containing HBSS prior to cutting.

A sheet of backing material was placed on a cutting board and the amnion was placed flat, epithelial side up, onto the backing material. A cutting apparatus having a cutting blade was used to cut the amnion and backing material together to create 5 cm×5 cm viable amnion tissue samples.

Each sample was placed into a retainer and then into a film pouch. The pouch was filled with 20-25 mL of cryopreservation solution comprising a basal medium and DMSO (10% final v/v) and sealed. The pouch was placed into an outer Tyvek pouch and sealed. The viable amnion tissue samples were then cryopreserved through a controlled-rate freezing method and stored at −70° C. or colder.

The viable amnion tissue samples were then subjected to thawing and equilibration, followed by enzymatic tissue digestion, and live/dead staining and cell count, as described in the Methodology section above. The live/dead working solution used for testing these viable amnion samples was obtained from a live/dead viability kit (LIVE/DEAD® Viability/Cytotoxicity Kit, Invitrogen), as described in the Methodology section section above.

A total of 45 images were processed and quantified for each of the three donors. All three evaluated donors had an average cell viability exceeding 70% (Table 1). These values suggest that cell viability of viable amnion membrane is well maintained after processing and cryopreservation.

TABLE 1 CELL VIABILITY DONOR 1 >70% DONOR 2 >70% DONOR 3 >70%

Example 2

The total cell count for three categories of viable amnion membrane, derived from multiple donors, were investigated by histological analysis. Representative samples of (A) native unprocessed amnion membrane, (B) processed and cryopreserved viable amnion membrane according to the processes described herein, and (C1) & (C2) processed and cryopreserved viable amnion according to a comparative process, as described in further detail below, were tested according to the methods described herein. Reported cell counts were then extrapolated for an approximate cell count per 5 cm×5 cm area and compared between the three categories of viable amnion membranes to determine whether processed viable amnion membranes contained a similar number of cells as native unprocessed amnion.

A. Unprocessed Viable Amnion Membrane (Comparative)

Samples of unprocessed amnion were collected from six donors immediately following separation of the amnion and chorion membranes, and prior to processing and cryopreservation. Each amnion sample was fixed in 10% neutral buffered formalin overnight or longer and then transferred into 70% ethanol for storage and shipping.

The average cell count values for all six samples, as determined by the laboratory, were averaged together to provide an average reported cell count which is shown in each of FIGS. 2 and 3.

B. Processed and Cryopreserved Viable Amnion Membrane

Samples of cryopreserved viable amnion membrane obtained from six different donors were produced by the following process, which was according to an embodiment of the method described and disclosed herein.

The viable amnion tissue samples were prepared as follows. For each donor, a fresh placenta was obtained. The amnion membrane was manually separated from the chorion and cut free from the placenta.

The amnion was placed into a first basin with HBSS for 5-10 minutes and manually cleaned during this period with wetted wipes to remove blood and blood clots. The amnion was transferred into a second basin with HBSS for 10-15 minutes and manually cleaned during this period with wetted wipes to remove blood and blood clots.

The amnion was then transferred into a flask containing antibiotic solution comprising vancomycin (50 μg/mL), gentamicin (50 μg/mL), amphotericin B (2.5 μg/mL), and HBSS. The flask was placed on an orbital shaker and agitated at 65 rpm for 60 minutes.

The amnion was then transferred to a second flask containing HBSS, placed onto an orbital shaker, and agitated at 65 rpm for 5 minutes. The amnion was then transferred to a third flask containing HBSS, placed onto an orbital shaker, and agitated at 65 rpm for 5 minutes. The amnion was then transferred to a holding basin containing HBSS prior to cutting.

A sheet of backing material was placed on a cutting board and the amnion was placed flat, epithelial side up, onto the backing material. A cutting apparatus was used to cut the amnion and backing material together to create 2 cm×2 cm viable amnion tissue samples.

Each sample was placed into a retainer and then into a film pouch. The pouch was filled with 20-25 mL of cryopreservation solution comprising a basal medium and DMSO (10% final v/v) and sealed. The pouch was placed into an outer Tyvek pouch and sealed. The viable amnion tissue samples were then cryopreserved through a controlled-rate freezing method and stored at −70° C. or colder.

The processed samples of cryopreserved viable amnion membrane were thawed per the package insert procedure in a water bath and rinsed in 5% dextrose in lactated ringer's (D5LR) solution to remove cryoprotectant. The samples were then fixed in 10% neutral buffered formalin overnight or longer and then transferred into 70% ethanol for storage and shipping.

The average cell count values for all of the samples, as determined by the laboratory, were averaged together to provide an average reported cell count which is shown in each of FIGS. 2 and 3.

C1 & C2. Processed and Cryopreserved Viable Amnion Membrane (Comparative)

For additional comparison, a first unit of viable amnion membrane (Grafix PRIME®, Osiris Therapeutics, Inc.) (C1) which was derived from a single donor and processed and cryopreserved by Osiris Therapeutics, Inc., was thawed and rinsed per package insert instructions. Samples of the tissue were cut and then fixed in 10% neutral buffered formalin overnight or longer and then transferred into 70% ethanol for storage and shipping. The average cell count value for this one sample (C1) is the value shown in FIG. 2.

In addition, two more units of viable amnion membrane (Grafix PRIME®, Osiris Therapeutics, Inc.) (C2) derived from two different donors and processed and cryopreserved by Osiris Therapeutics, Inc., was thawed and rinsed per package insert instructions. Samples of the tissue were cut and then fixed in 10% neutral buffered formalin overnight or longer and then transferred into 70% ethanol for storage and shipping. The average cell count values for the one earlier sample (C1) and the two new samples (C2), as determined by the laboratory, were averaged together to provide an average reported cell count which is shown in each of FIG. 3.

Testing

For all three categories of viable amnion membranes (A), (B) and (C1)/(C2), fixed samples were sent to a histology lab for paraffin embedding, sectioning, and H&E staining with Mayer's Hematoxylin solution and Eosin Y solution. Prepared H&E slides were examined manually for cell nuclei stained by the hematoxylin solution and the epithelial and stromal layers were counted separately in 12 random selected areas for each sample. Average cell density and standard deviation was reported for each sample. The average cell density was then extrapolated to a cell count for a 5 cm×5 cm area of tissue for each donor. Results were then averaged for unprocessed amnion membrane, viable amnion membrane, or competitor viable amnion membrane donors.

Results

As shown in FIG. 2, the processed and cryopreserved viable amnion membrane (A) according to an exemplary embodiment of the process described herein contained a similar number of cells as unprocessed amnion membrane (B). This suggests that when viable amnion membrane is processed, cryopreserved, and prepared according to the present invention, the number of native epithelial mesenchymal cells initially present in the amnion membrane is not significantly impacted.

Additionally, as shown in FIG. 2, the first sample of processed and cryopreserved viable amnion membrane (C1) according to a comparative process (i.e., Grafix PRIME® by Osiris Therapeutics, Inc.) had an average calculated cell count of approximately 47% of the number of cells contained in the unprocessed amnion membrane, which indicates that amnion cells are lost at some portion of the processes performed by Osiris Therapeutics, Inc. As shown in FIG. 3, the expanded set of samples of processed and cryopreserved viable amnion membrane (C2) according to the comparative process (i.e., Grafix PRIME® by Osiris Therapeutics, Inc.) had an average calculated cell count of approximately 72% of the number of cells contained in the unprocessed amnion membrane, which also indicates that amnion cells are lost at some portion of the processes performed by Osiris Therapeutics, Inc.

As shown in FIG. 4, representative H&E images of the unprocessed and differently processed viable amnion membranes shows differences in the populations of epithelial, amnion, and mesenchymal cells. More particularly, image (a) shows H&E stained unprocessed amnion, image (b) H&E stained viable amnion according to an exemplary embodiment of the process described herein, and image (c) shows H&E stained viable amnion membrane according to a comparative process (i.e., Grafix PRIME® by Osiris Therapeutics, Inc.). The downward pointing arrows in each of images (a), (b) and (c) indicate the epithelial side of each viable amnion and the epithelial cells thereon. Similarly, the upward pointing arrows in each of images (a), (b) and (c) indicate the stromal side of each amnion membrane and the amnion and mesenchymal cells embedded therein.

The difference in the populations of cells in each of the viable amnion membranes (A), (B), (C) can also be seen in the H&E stained images shown in FIG. 4. More particularly, the unprocessed amnion membrane (A) and the processed and cryopreserved viable amnion membrane according to the present invention (B) both show relatively intact layers of epithelial cells on the epithelial side of the amnion and mesenchymal stromal cells interspersed within the stromal layer on the stromal side of the amnion (images (a) and (b)). In contrast, the processed and cryopreserved viable amnion membrane (i.e., Grafix PRIME®) according to the comparative process by Osiris Therapeutics, Inc. has a noticeably sparser epithelial layer (image (c)).

Example 3

The differentiation capability of cells on intact viable amnion membrane into osteogenic and chondrogenic lineages was investigated. Samples of viable amnion membrane were cultured in osteogenic and chondrogenic differentiation media for up to 8 weeks. Samples were fixed in 10% neutral buffered formalin and then sent for special histological staining. Osteogenic differentiation samples were stained with von Kossa and Alizarin Red S, and chondrogenic differentiation samples were stained with Alcian Blue. These were then evaluated for staining indicative of successful differentiation. Positive Alizarin Red, von Kossa, and Alcian Blue staining showed the continued differentiation capability of amnion cells that were retained on the allograft after processing and cryopreservation.

Samples of cryopreserved viable amnion membrane were thawed per the package insert procedure in a room temperature water bath and rinsed in 5% dextrose in lactated ringer's (D5LR) solution to remove cryoprotectant. The samples were placed into cell culture media to allow post-thaw equilibration before switching to osteogenic, or chondrogenic differentiation media for test samples or continuing to culture in cell culture media as negative controls. Samples of viable amnion membrane were cultured for 2, 4, or 8 weeks in the differentiation media, or in cell culture media (supplemented basal media without differentiation signaling cues) serving as negative controls, with media changes twice a week.

At each planned time point, tissue samples in cell culture media and in various differentiation media were collected and fixed in 10% neutral buffered formalin overnight. Fixed amnion samples were sent to a histology lab for paraffin embedding, sectioning, and staining with von Kossa and Alizarin Red S (osteogenic) or Alcian Blue (chondrogenic) stains as appropriate. Prepared slides of samples cultured in differentiation media were examined using an upright microscope (Olympus, Waltham, MA) to evaluate positive staining indicative of cells in the amnion tissue undergoing successful differentiation as compared to the negative control samples stained with the same.

FIG. 5 provides images of positive Alizarin Red S stained (red stain) representative viable amnion membrane samples after culture in osteogenic media for 2 weeks (A), 4 weeks (B), and 8 weeks (C), showing calcium deposition. FIG. 5 also provides images of positive von Kossa stained (dark brown/black stain) representative viable amnion membrane tissue cultured in osteogenic media for 2 weeks (D), 4 weeks (E), and 8 weeks (F), and also show mineralization of the amnion. Images of negative control amnion membranes cultured in cell culture media showing no positive Alizarin Red S (G) and von Kossa (H) staining.

Osteogenic differentiation of the viable amnion membrane was visualized via Alizarin Red S and von Kossa staining (FIG. 5). Alizarin Red S forms an Alizarin Red S-calcium complex in a chelation process, producing a red stain. The von Kossa stain is a precipitation reaction in which silver ions replace calcium under strong light and form a brown or black deposit in the presence of calcium or calcium salts. Both stains, as shown in FIG. 5, indicate mineralization of viable amnion membrane cultured in osteogenic differentiation media occurring as early as 2 weeks, with increasing intensity over time up to 8 weeks. In contrast, negative controls did not exhibit either Alizarin Red or von Kossa staining at 8 weeks. These findings demonstrate the ability of amnion cells on intact tissue to differentiate into an osteogenic lineage.

FIG. 6 provides images of Alcian Blue stained (blue stain) representative viable amnion membrane samples after culture in chondrogenic media for 2 weeks (A), 4 weeks (B), and 8 weeks (C), show strong presence of GAGs by 8 weeks. FIG. 6 also provides an image of negative control amnion membrane cultured in cell culture media showing no significant Alcian Blue staining at 8 weeks (D).

Chondrogenic differentiation of the viable amnion membrane was visualized via Alcian Blue staining (FIG. 6). Alcian Blue is used to stain mucosubstances and proteoglycans such as glycosaminoglycans (GAGs) in cartilage a vivid blue color. At 8weeks, the amnion membrane appears to be undergoing chondrogenic differentiation as shown by the intense Alcian Blue staining as compared to negative control and also as compared to the 2 and 4 week time point samples. There is some lighter blue stain color outside the tissue area in the 2 and 4 week time point samples but are attributed to artifacts of the embedding and slide preparation process, and is not considered positive staining in the tissue samples themselves. The positive Alcian Blue staining indicates the ability of amnion cells on intact amnion to differentiate into a chondrogenic lineage.

The cells on the intact tissue were shown to be capable of undergoing osteogenic and chondrogenic differentiation over time. This data demonstrates that the method described herein for processing of viable amnion membrane, followed by cryopreservation, retains functional progenitor cells in the tissue. These cells, in addition to endogenous matrix proteins, growth factors, and cytokines are key biological components in an amnion graft that can support the progression of the healing process. 

1. A method for producing a tissue graft comprising at least one processed tissue fragment derived from naturally occurring unprocessed membranous tissue, the method comprising: obtaining the unprocessed membranous tissue comprising amnion membrane which has an unprocessed population size of endogenous cells; and subjecting the unprocessed membranous tissue to one or more processes selected from: a cleaning process, a soaking process, a bioburden reducing process, an additional soaking process, a rinsing process, and a freezing process, wherein at least one or more of the processes is performed by contacting the unprocessed membraneous tissue with one or more solutions, wherein none of the solutions comprises an anticoagulant, thereby producing the at least one processed tissue fragment, which has a retained population size of endogenous cells which is at least 90 percent of the unprocessed population size, and at least 70 percent of the retained population size of endogenous cells are viable.
 2. The method for producing a tissue graft of claim 1, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 3. The method for producing a tissue graft of claim 2, wherein the one or more monosaccharides comprises at least one of: glucose, fructose, and galactose.
 4. The method for producing a tissue graft of claim 1, wherein the bioburden reducing process comprises contacting the unprocessed membraneous tissue with an antibiotic solution for a short period of time which is less than about 60 minutes.
 5. The method for producing a tissue graft of claim 4, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 6. The method for producing a tissue graft of claim 4, wherein the freezing process comprises a controlled rate freezing process.
 7. The method for producing a tissue graft of claim 6, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 8. The method for producing a tissue graft of claim 1, wherein the freezing process comprises a controlled rate freezing process.
 9. The method for producing a tissue graft of claim 8, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 10. The method for producing a tissue graft of claim 1, wherein the freezing process comprises contacting the unprocessed membranous tissue with a cryopreservation solution which lacks serum.
 11. The method for producing a tissue graft of claim 10, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 12. The method for producing a tissue graft of claim 10, wherein the freezing process comprises a controlled rate freezing process.
 13. The method for producing a tissue graft of claim 12, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 14. The method for producing a tissue graft of claim 1, wherein the at least one processed tissue fragment comprises either: at least one sheet, each having a shape with an average length of about 1 to about 20 centimeters (cm) and an average width of about 1 to about 20 cm, or at least one mini sheet, each having a shape with an average length of about 0.5 to about 9 mm and an average width of about 0.5 to about 9 mm.
 15. The method for producing a tissue graft of claim 14, wherein the at least one processed tissue fragment comprises at least one sheet, each having a shape with an average length of about 1 to about 10 centimeters (cm) and an average width of about 1 to about 10 cm.
 16. A method for producing a tissue graft comprising at least one processed tissue fragment derived from naturally occurring unprocessed membranous tissue, the method comprising: obtaining the unprocessed membranous tissue comprising amnion membrane which has an unprocessed population size of endogenous cells; and subjecting the unprocessed membranous tissue to one or more processes selected from: a cleaning process, a soaking process, a bioburden reducing process, an additional soaking process, a rinsing process, and a freezing process, wherein at least one or more of the processes is performed by contacting the unprocessed membraneous tissue with one or more solutions, wherein the bioburden reducing process comprises contacting the unprocessed membraneous tissue with an antibiotic solution for a short period of time which is less than about 60 minutes, thereby producing the at least one processed tissue fragment, which has a retained population size of endogenous cells which is at least 90 percent of the unprocessed population size, and at least 70 percent of the retained population size of endogenous cells are viable.
 17. The method for producing a tissue graft of claim 16, wherein the short period of time is less than about 30 minutes.
 18. The method for producing a tissue graft of claim 16, wherein the short period of time is less than about 10 minutes.
 19. The method for producing a tissue graft of claim 16, wherein the short period of time is from about 1 minute to less than about 5 minutes.
 20. The method for producing a tissue graft of claim 16, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 21. The method for producing a tissue graft of claim 20, wherein the one or more monosaccharides comprises at least one of: glucose, fructose, and galactose.
 22. The method for producing a tissue graft of claim 16, wherein the freezing process comprises a controlled rate freezing process.
 23. The method for producing a tissue graft of claim 22, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 24. The method for producing a tissue graft of claim 16, wherein the freezing process comprises contacting the unprocessed membranous tissue with a cryopreservation solution which lacks serum.
 25. The method for producing a tissue graft of claim 24 wherein at least one of the one or more solutions comprises one or more monosaccharides.
 26. The method for producing a tissue graft of claim 24, wherein the freezing process comprises a controlled rate freezing process.
 27. The method for producing a tissue graft of claim 26, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 28. The method for producing a tissue graft of claim 16, wherein the at least one processed tissue fragment comprises either: at least one sheet, each having a shape with an average length of about 1 to about 20 centimeters (cm) and an average width of about 1 to about 20 cm, or at least one mini sheet, each having a shape with an average length of about 0.5 to about 9 mm and an average width of about 0.5 to about 9 mm.
 29. The method for producing a tissue graft of claim 28, wherein the at least one processed tissue fragment comprises at least one sheet, each having a shape with an average length of about 1 to about 10 centimeters (cm) and an average width of about 1 to about 10 cm.
 30. A method for producing a tissue graft comprising at least one processed tissue fragment derived from naturally occurring unprocessed membranous tissue, the method comprising: obtaining the unprocessed membranous tissue comprising amnion membrane which has an unprocessed population size of endogenous cells; subjecting the unprocessed membranous tissue to one or more processes selected from: a cleaning process, a soaking process, a bioburden reducing process, an additional soaking process, a rinsing process, and a freezing process, wherein at least one or more of the processes is performed by contacting the unprocessed membraneous tissue with one or more solutions, wherein the freezing process comprises contacting the unprocessed membranous tissue with a cryopreservation solution which lacks serum, thereby producing the at least one processed tissue fragment, which has a retained population size of endogenous cells which is at least 90 percent of the unprocessed population size, and at least 70 percent of the retained population size of endogenous cells are viable.
 31. The method for producing a tissue graft of claim 30, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 32. The method for producing a tissue graft of claim 31, wherein the one or more monosaccharides comprises at least one of: glucose, fructose, and galactose.
 33. The method for producing a tissue graft of claim 30, wherein the freezing process comprises a controlled rate freezing process.
 34. The method for producing a tissue graft of claim 33, wherein at least one of the one or more solutions comprises one or more monosaccharides.
 35. The method for producing a tissue graft of claim 30, wherein the at least one processed tissue fragment comprises either: at least one sheet, each having a shape with an average length of about 1 to about 20 centimeters (cm) and an average width of about 1 to about 20 cm, or at least one mini sheet, each having a shape with an average length of about 0.5 to about 9 mm and an average width of about 0.5 to about 9 mm.
 36. The method for producing a tissue graft of claim 35, wherein the at least one processed tissue fragment comprises at least one sheet, each having a shape with an average length of about 1 to about 10 centimeters (cm) and an average width of about 1 to about 10 cm.
 37. A method for producing a tissue graft comprising at least one processed tissue fragment derived from naturally occurring unprocessed membranous tissue, the method comprising: obtaining the unprocessed membranous tissue comprising amnion membrane which has an unprocessed population size of endogenous cells and a bioburden; cleaning the unprocessed membranous tissue by manually manipulating the unprocessed membranous tissue to at least remove blood clots; performing a first soak by contacting the unprocessed membranous tissue with a first isotonic salt solution for a first period of time of from about 10 to about 20 minutes, optionally performing a second soak by draining the isotonic salt solution from the membranous tissue and contacting the membranous tissue with a second isotonic salt solution for a second period of time of from about 10 to about 20 minutes; reducing the bioburden of the membranous tissue by contacting the membranous tissue with an antibiotic solution comprising an antibiotic for a period of time of at least about 30 minutes and less than about 60 minutes; performing one or more additional soaks, after reducing the bioburden, to rinse the antibiotic solution away from the membranous tissue by contacting the membranous tissue at least once with a third isotonic salt solution for at least a third period of time of from about 5 to about 10 minutes and contacting the membranous tissue with a fourth isotonic salt solution for at least a fourth period of time of from about 30 to about 40 minutes, thereby producing a processed membranous tissue; producing the at least one processed membranous tissue fragment by mechanically cutting the membranous tissue with a device having one or more blades; optionally placing the at least one processed membranous tissue fragment into a packaging and filling the packaging with cryopreservation solution, wherein no serum is included in the cryopreservation solution; and optionally freezing the at least one processed membranous tissue fragment by controlled rate freezing to −80° C. or less, wherein the freezing step is initiated within about 8 hours after completion of the cleaning step and within about 1 hour after the filling with cryopreservation solution step is performed; wherein each of the first, second, third, and fourth isotonic salt solutions is, independently, the same or different from the other isotonic salt solutions; wherein no anticoagulant is included in any of the first, second, third, or fourth isotonic salt solutions, the antibiotic solution, or the cryopreservation solution; and wherein one or more of the buffer and antibiotic solutions comprises a monosaccharide comprising glucose, such that the at least one processed tissue fragment has a retained population size of endogenous cells which is at least 90 percent of the unprocessed population size, and at least 70 percent of the retained population size of endogenous cells are viable. 